From solid medium, strains were grown aerobically in a rotary shaker at 37°C and 160 rpm in YEC, a liquid yeast extract medium supplemented with L-cysteine, ferric pyrophosphate and α-ketoglutarate. Overnight cultures were diluted 100-fold in YEC liquid medium and were allowed to grow for 24 h (to the stationary phase). Biocides challenge Stationary phase L. pneumophila cells were harvested from 250 ml cultures and then washed twice with phosphate buffer (pH 7.4) by centrifugation at 5500 × g for

10 min at 4°C. Cells were resuspended and diluted in PBS pH 7.4 to an optical density of 0.2 at 600 nm (1 × 108 cells ml-1). These cell suspensions (50 ml each) were distributed into 100-ml glass flasks, and fresh HOCl solution (prepared the same day) was added to various concentrations from 0 to 1 mM (≤1 ml). The samples were incubated www.selleckchem.com/products/btsa1.html Cilengitide price for 1 h at 37°C in the dark with agitation (160 rpm), and the HOCl was then quenched by the addition of sterile sodium thiosulfate (final concentration 0.4 mM). Culturable bacteria were assayed by plating serial dilutions in PBS on BCYE plates at 37°C. KPT-8602 Colonies were counted after 3 days and 10 days of incubation at 37°C. Viability staining procedure Viability of L. pneumophila was assessed using the ChemChrome V6 procedure (ChemChrome V6; CV6 – AES-Chemunex, Ivry-sur-seine, France) and the total number of cells was assessed using the DAPI

procedure (DAPI Nucleic Acid Stain; Invitrogen). Aliquots of a suspension of 1 × 107 cells ml-1were used for staining experiments. Labeling

solutions were added to the samples Acetophenone according to the manufacturer’s instructions and incubated at 37°C for 30 min in the dark; they were washed by centrifugation (4500 × g for 10 min in PBS pH 7.4) and transferred into 96-well glass-bottom micro plates (Greiner Bio One) previously treated with poly-L-lysine (0.01%). To favor cell adhesion to the wells, the plates were centrifuged at 1000 × g for 20 min. For each condition, the number of viable cells and the total number of cells were counted, and the results reported are mean values for three independent wells in which at least 3000 cells were analyzed. The distribution of the normalized fluorescent intensity of cells as detected by microscopy is presented as histograms. The values 0 and 1 represent the maximum and the minimum values, respectively, of fluorescence observed in all tested conditions for each experiment. The proportion of each class were normalized to the number of viable cells. The Mann–Whitney U test was used to assess the significance of differences in viable, total and culturable cell numbers. Fluorescence microscopy Microscopic analyses were performed using the automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon) with the appropriate filter blocks as previously described [47].